【作者】 魏宝阳；

【导师】 李顺祥； 卢向阳；

【作者基本信息】 湖南农业大学 ， 生物化学及分子生物学， 2012， 博士

【摘要】 [目的]贵州铜仁地区产的吴茱萸,颗粒均匀、色泽绿、香气浓烈、味辛辣、品质上乘,饮誉中外。因贵州铜仁地区所产吴茱萸产量大、质量优良,被称为贵州道地药材,也为贵州名药之一；湖南新晃产吴莱萸质量最好,为吴茱萸道地药材。研究中药吴茱萸道地性形成的相关因子,探讨中药吴茱萸道地性形成的机制,构建吴茱萸道地药材产地适宜性分析评价的共性技术体系,结合社会经济条件,为因地制宜地进行生产区域布局和发展优势品种提供技术支撑,促进吴茱萸道地药材的可持续发展。[方法]通过网络调查了全国吴茱萸资源；实地考察了全国吴茱萸主要产区并对产区的种植品种、引种来源、蕴含量、经纬度、海拔、土壤类型等进行详细记录,采用五点采样法采集这些产区的根、茎、叶、果实和土壤等样品；利用TCMGIS技术对中药吴茱萸道地产区进行区划分析；用吴茱萸的嫩叶为试验材料,利用ITS、AFLP、SRAP等分子标记技术,对不同产地的吴茱萸进行遗传背景分析；采用平板分离法对湖南新晃、浏阳、湘乡、娄底,贵州铜仁,江西万载和浙江平阳、建德等地的吴茱萸内生真菌进行了分离纯化及比较分析,采用HPLC, LC-MS等分析技术对产吴茱萸碱、吴茱萸碱次碱及结构类似物等成分的微生物进行分析；采用HPLC对吴茱萸碱和吴茱萸次碱的含量进行测定,采用原子吸收法测定中药吴茱萸果实中微量元素的含量；采用滴定法测定土壤呼吸强度、采用最大或然计数法(MPN)进行微生物生理类群(钾细菌、氨化细菌、自生好氧固氮菌、硝化细菌、纤维素分解菌)的测定,采用比色法测定土壤酶脲酶、蔗糖酶、过氧化氢酶和蛋白酶等酶的活性,采用原子吸收法测定土壤微量元素的含量。[结果]1.文献查阅和实地考察发现,中药吴茱萸的道地性与纬度(27。左右)和海拔(200-600m)有关,说明纬度和海拔是中药吴茱萸道地性形成的关键因子；通过吴茱萸的生物学特性与产量的关系分析可以看出,吴茱萸品种的选择对吴茱萸的品质有很大的影响；通过TCMGIS的吴茱萸产地适宜性区划分析发现吴茱萸中的石虎品种82%的适宜产地在贵州,贵州作为石虎的道地产区与文献记载的完全吻合。2.ITS分析结果表明,吴茱萸药材ITS序列多态性较差,序列相似度大,仅部分样品存在少量碱基突变；AFLP分析结果表明,AFLP分子标记技术能很好地区分吴茱萸药材三个来源植物吴茱萸和石虎、疏毛吴茱萸之间的遗传差异；湖南新晃、贵州铜仁等高海拔山区作为中药吴茱萸道地产区,植株遗传背景比较稳定,这可能是生产质量稳定的药材原料的基础；SRAP分析结果表明,吴茱萸遗传背景多样性比较明显,由SRAP对吴茱萸的聚类分析可知,吴茱萸遗传背景有品种的差异,即石虎和疏毛吴茱萸的遗传差异较大,各地区有按同一品种聚类的趋势,同一品种不同产地的遗传背景差异较小,突出表现为贵州铜仁、湖南新晃道地产区的样品聚集较紧密。3.对不同产地同一品种、同一产地不同品种、同一产地同一品种的不同采收期的吴茱萸碱和吴茱萸次碱的含量检测表明,品种、产地、采收期对中药吴茱萸的道地性都有重要的影响。4.对不同吴茱萸产区的土壤因子研究表明,土壤Zn、土壤蔗糖酶活、土壤Cu等因子对中药吴茱萸道地性有显著影响。5.从8个吴茱萸产区共分离到31株内生真菌和37株内生细菌,分别从吴茱萸的叶、茎和果实中分离到不同的内生菌,表现为组织特异性,且在不同产地的内生菌的种类和优势种群存在明显的差异,即具有地域特异性；筛选出产吴茱萸碱和次碱及结构类似物的4株内生真菌和1株内生细菌。6.吴茱萸挥发油的抑菌试验表明,吴茱萸挥发油的抑菌能力可以作为中药吴茱萸道地性的一个重要的药效指标。[结论]中药吴茱萸道地性的形成主要是以下因子共同作用的结果：各产地吴茱萸栽培品种的差异、遗传背景的特异性等内在因素起决定性作用,如品质最好的湖南新晃的中药吴茱萸品种单一,全为石虎,其遗传背景差异小且稳定；环境因子中的温度,海拔和土壤微生物的总量和分布规律以及土壤元素的分布规律等外在因素起关键作用。研究吴茱萸道地性的技术包括：用于遗传背景分析的AFLP和SRAP等分子标记技术,用于道地产地区划的TCMGIS-Ⅱ系统分析法,用于土壤微生物的生理类群分析的最大或然法,用于土壤总微生物的含量测定的酸碱滴定法。更多还原

【Abstract】 [Objective]Euodia ruticarpa of Tongren Guizhou is popular at home and abroad because uniform particles, green colour and lustre, strong fragrance, acridity sapor and excellent quality. With high yield and good quality, Tongren Guizhou produces Euodia ruticarpa usually is looked as genuine medicinal materials of Guizhou and one of Guizhou famous medicinal materials, too. The Euodia ruticarpa of Hunan Xinhuang is also genuine medicinal materials for the best quality. In order to establish generic technology system of appraiseing Euodia rutaecarpa of adaptive region, the correlation factors and forming mechanism of Euodia rutaecarpa genuine were researched. Combing with social and economic conditions, the research would not only to provide technical support for producing region composition according to the local conditions and developing advantage varieties, but also to promote the sustainable development of superior medicinal materials of Euodia rutaecarpa.[Methods]Euodia rutaecarpa resource was investigated by network. The main producing areas of Euodia rutaecarpa was investigated via site visits. Plant varieties, introduction, yields, latitudes and longitudes, elevation and soil types were recorded in the main producing areas. Euodia rutaecarpa samples including roots, stems, fruits and soil were collected using five sampling method. The regionalization of Euodia rutaecarpa genuine region was analysed using TCMGIS technology. The genetic diversity of Euodia rutaecarpa with fresh leaves was studied using ITS, SRAP, AFLP molecular markers. Using plate method, the Endophytes in Euodia rutaecarpa were separated and purified and compared in Xinhuang, Liuyang, Xiangxiang, Luodi of Hunan, Tongren Guizhou, Wanzhai Jiangxi and Pingyang, Jiangde Zhejiang. Using HPLC, LC-MS technology, the components about evodiamine, rutaecarpine and structural analogues were analyzed. HPLC method was used to detect the content of Euodiamine and Rutaecarpine in Euodia rutaecarpa. The content of the trace element in Euodia rutaecarpa was determined by atom absorption. Respiration of soil microbe was studied by titration analytical procedures; the microbial physiological groups (decomposing Potassium bacterium, aminate bacteria, Azotobacter, nitrifying bacteria, cellulose decomposition bacteria)in soils were tested by most probable number. Soil enzymatic activities(urease, catalase, sucrase, Proteinase)were determined by colorimetry. The content of the trace element in soil was determined by atom absorption.[Results]1. The result of literature review and investigation showed that the genuineness of Euodia rutaecarpa was associated with the latitude(about27°) and altitude(200～600m). the latitude and altitude were key factors of forming the genuineness of Euodia rutaecarpa. Variety influenced the genuineness of Euodia rutaecarpa distinctly. Using TCMGIS, suitable growth regions was analysed, the resulted showed that82%genuineness regions of E. Benth.var.oficinalis(Dode)Huang located in Guizhou, witch was Completely consistent with records.2. The result of ITS analysis showed that ITS sequence polymorphism of Fructus Euodia was poorer, the sequence was similar, only some samples contain few base mutations.The result of AFLP analysis showed that it was easy to distinguish the genetic diversity of three Medicinal Materials source plants Euodia rutaecarpa, Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang and Euodia rutaecarpa(Juss) Benth var. bodinieri (Dode) Huang. Plant genetic background was stable in High altitude mountain areas, which are Euodia rutaecarpa authentic region such as Xinhuang of Hunan, Tongren of Guizhou etc. This would be the foundation production quality stable medicinal material. The result of SRAP analysis showed that Euodia rutaecarpa genetic background diversity was obvious. Using cluster analysis of SRAP, we concluded that there were varieties differences in Euodia rutaecarpa genetic background. The genetic differences were obvious between Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang and Euodia rutaecarpa(Juss) Benth var.bodinieri (Dode) Huang, and there was a trend of same variety to clustering in each region. Obviously, the genetic background of the same variety from different origin, those samples gathered more closely from Euodia rutaecarpa authentic region such as Xinhuang of Hunan, Tongren of Guizhou.3. Content of Euodiamine and Rutaecarpine in different producing areas of the same variety, the same origin of different varieties, the same variety of different harvest time showed that varieties, origin and harvest time influenced on the genuineness of Euodia rutaecarpa distinctly.4. The results of soil factor showed that Zn, sucrase, Cu influenced on the genuineness of Euodia rutaecarpa significantly.5.31endophytic fungi and37endophytic bacteria in Euodia rutaecarpa were separated from8Euodia rutaecarpa region, different specific types of the endophytic fungi and bacteria were separated in different leaves, stems, and fruits, namely with organized specificity. Existing obvious differences in different types of the Endophytes and dominant population, namely with regional specificity.4Euodia rutaecarpa endophytic fungi and1endophytic bacteria were screened which could produce evodiamine, rutaecarpine and structural analogues.6. The result of bacteriostatic experiment on Euodia rutaecarpa volatile oil showed that the bacteriostatic ability of Euodia rutaecarpa volatile oil could be regarded as an important efficacy index of Euodia rutaecarpa genuineness.[Conclusion]Influencing factors of forming genuineness of traditional Chinese medicine Euodia rutaecarpa include internal factors and Extrinsic Factors. Internal factors played decisive role on the formation of genuineness such as the variety difference of Euodia rutaecarpa and the specificity of the genetic background. For example, Euodia rutaecarpa of Xinhuang Hunan was the only variety of Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang, the genetic background was little difference and stable. Extrinsic Factors included temperature, altitude, the amount and distribution law of soil microorganisms, distribution law of trace elements in soil, witch played key role. The following technology used to research genuineness of Euodia rutaecarpa:AFLP and SRAP molecular markers used for genetic background analysis, TCMGIS-Ⅱ system analysis for genuine origin division, most probable number was applied to the analysis of soil microorganism physiological groups, acid-base titration method was applied to the total content determination of soil microorganisms.更多还原